Study On The Mechanism Of Shenqi Fuzheng Injection In The Intervention Of Gastric Cancer Based On Evidence-based Medicine Combined With Network Pharmacology

Objective:Utilizing technologies such as evidence-based medicine,network pharmacology,and molecular docking,along with in vitro and in vivo experiments,we investigate the clinical impacts and molecular pathways involved in the use of Shenqi Fuzheng Injection for gastric cancer treatment.This study provides scientific basis for further study of the mechanism of Shenqi Fuzheng Injection in the treatment of gastric cancer and clinical medication.Methods:（1）A overview of systematic reviews and re-evaluation study analyzed the clinical data of Shenqi Fuzheng Injection for treating malignant tumors.Randomized controlled trials were independently screened from systematic reviews and Meta-analyses on Shenqi Fuzheng Injection for gastric cancer treatment,and relevant data were extracted.The risk of bias in the included studies was assessed,and Meta-analysis was performed using RevMan 5.4 software.（2）Network pharmacology research was used to screen the key targets of Shenqi Fuzheng Injection and gastric cancer,molecular docking technology was used to verify the binding ability of key targets and main compounds.Screen key signaling pathways through GO functional enrichment and KEGG pathway enrichment.（3）Taking human gastric cancer cells MKN45 and AGS cells as the research subjects,after drug intervention with Shenqi Fuzheng Injection,CCK-8 method was used to detect cell viability,EDU experiment was used to detect the level of proliferation,and flow cytometry was used to detect the level of apoptosis.RT-PCR experiments were used to detect the proliferation-related genes PCNA and Ki67,apoptosis-related genes Bax,Bcl-2,Caspase3,Caspase9 and PI3K/AKT/mTOR signaling pathway gene transcription levels,and WB experiments were used to detect the proliferation-related proteins PCNA and Ki67respectively.The expression levels of apoptosis-related proteins Bax,Bcl-2,Caspase3 and Caspase9 and PI3K/AKT/mTOR signaling pathway proteins were further verified by adding the PI3K/AKT/mTOR signaling pathway agonist IGF-1.（4）The experiment used 40 gastric cancer MKN45 cell tumor-bearing nude mice as the research subjects,and they were randomly divided into four groups:model group（Model）,Shenqi Fuzheng injection group（SQ）,cisplatin group（DDP）,Shenqi Fuzheng In the injection+cisplatin group（SQ+DDP）,after intraperitoneal injection intervention,tumor samples from mice in each group were obtained,and their tumor inhibition rates were calculated.WB experiments were performed to detect the proliferation-related proteins PCNA and Ki67 in model mouse tumor tissue,and apoptosis Expression levels of related proteins Bax,Bcl-2,Caspase3 and Caspase9 and PI3K/AKT/mTOR signaling pathway proteins.immunohistochemistry experiments detected PI3K/AKT/mTOR signaling pathway phosphorylated proteins.Results:（1）The results of Meta-analysis of gastric cancer systematic review re-evaluation showed that,.compared with the conventional chemotherapy group,Shenqi Fuzheng Injection combined with conventional chemotherapy could improve the clinical efficacy（RR=1.38,95%CI:1.27～1.50）and quality of life（RR=1.37,95%CI:1.23～1.52）of patients with gastric cancer.In terms of immune function and adverse reactions,Shenqi Fuzheng Injection combined with chemotherapy could increase the levels of CD3+and CD4+T cells（CD₃⁺:MD=10.59,95%CI:7.52～13.67;CD₄⁺:MD=8.30,95%CI:5.05～11.55）,and reduce the incidence of thrombocytopenia（RR=0.70,95%CI:0.58～0.85）,leukopenia（RR=0.73,95%CI:0.60～0.90）,renal impairment（RR=0.60,95%CI:0.36～0.97）,neurotoxicity（RR=0.60,95%CI:0.44～0.82）,nausea and vomiting（RR=0.58,95%CI:0.50～0.67）.（2）Network pharmacology research results show that TP53,ESR1,AKT1,JUN,MAPK3,EP300,MAPK1 and SRC may be the key targets of Shenqi Fuzheng Injection in the treatment of gastric cancer.Molecular docking research found that AKT1 and the core active ingredient compound the strongest binding ability,through GO functional enrichment and KEGG pathway enrichment,it was found that PI3K/AKT/mTOR,HIF-1 and IL-17 and other signaling pathways may be the key pathways for Shenqi Fuzheng Injection to treat gastric cancer.Among the eight key targets,the PI3K/AKT/mTOR signaling pathway is the most significantly enriched.The PI3K/AKT/mTOR（AKT1）signaling pathway may be the most critical signaling pathway for Shenqi Fuzheng Injection to treat gastric cancer.（3）In vitro experimental results:through the CCK8 experiment,MKN45 cells were screened with low（IC30）,medium（IC50）and high（IC70）intervention concentrations of 130mg/mL,150 mg/mL and 170 mg/mL respectively;AGS cells with low（IC30）,medium（IC50）)high（IC70）intervention concentrations were 60 mg/mL,80 mg/mL and 110 mg/mL respectively.The EDU experiment results:the number of positive cells in the Shenqi Fuzheng Injection intervention group decreased significantly（P<0.05）.The flow cytometry test results:the apoptosis rates of MKN45 and AGS cells increased significantly（P<0.05）.The RT-PCR test results:the PCNA,Ki67,Bcl-2 and gene transcription levels of the two groups of cell lines were significantly decreased,and the Bax,Caspase3 and Caspase9 gene transcription levels were significantly increased（P<0.05）,and the PI3K/AKT/mTOR signaling pathway PI3K,AKT and mTOR genes significant decrease（P<0.05）.WB detection results:PCNA,Ki67,Bcl-2 and protein expression of two groups of cell lines were significantly down-regulated,Bax,Caspase3 and Caspase9 protein expression were significantly up-regulated（P<0.05）,there were no significant changes in pathway PI3K,AKT and mTOR proteins of the PI3K/AKT/mTOR signaling pathway（P>0.05）,and the expression of pathway proteins p-PI3K,p-AKT and p-mTOR proteins was significantly down-regulated（P<0.05）.After adding the PI3K/AKT/mTOR signaling pathway agonist IGF-1,the expressions of PCNA,Ki67 and Bcl-2 proteins in the two groups of cell lines in the Shenqi Fuzheng Injection group were significantly down-regulated,while the expressions of Bax,Caspase3and Caspase9 were all up-regulated（P<0.05）.There were no significant changes in pathway PI3K,AKT and mTOR proteins of the PI3K/AKT/mTOR signaling pathway（P>0.05）,p-PI3K,p-AKT and p-mTOR protein expression was significantly down-regulated.the apoptosis rate of MKN45 and AGS cells,PCNA,Ki67 and Bcl-2 protein expression in the Shenqi Fuzheng Injection group were significantly higher than those in the SQ+IGF-1 group（P<0.05）.The protein expressions of Bax,Caspase3,Caspase9,there were no significant changes in pathway PI3K,AKT and mTOR proteins of the PI3K/AKT/mTOR signaling pathway（P>0.05）,p-PI3K,p-AKT,and p-mTOR in the Shenqi Fuzheng Injection group were lower than those in the Shenqi Fuzheng Injection+IGF-1 group,and the differences were statistically significant（P<0.05）.Compared with the Shenqi Fuzheng Injection+IGF-1 group the apoptosis rate of MKN45 and AGS cells,Bax,Caspase3,and Caspase9 protein expression in the Shenqi Fuzheng Injection group were significantly higher than those in the Shenqi Fuzheng Injection+IGF-1 group（P<0.05）.The protein expressions of PCNA,Ki67,Bcl-2,p-PI3K,p-AKT and p-mTOR in the Shenqi Fuzheng injection group were lower than those in the Shenqi Fuzheng injection+IGF-1 group,and the differences were statistically significant scientific significance（P<0.05）.（4）In vivo experimental results:The tumor mass of the Shenqi Fuzheng injection group,cisplatin group,and Shenqi Fuzheng injection+cisplatin group was significantly reduced,and the difference was statistically significant（P<0.05）.The tumor inhibition rates of the Shenqi Fuzheng Injection group,the cisplatin group,and the Shenqi Fuzheng Injection+cisplatin group were 30%,36%,and 52%respectively.Compared with the model group,the protein expressions of PCNA,Ki67 and Bcl-2 in the Shenqi Fuzheng Injection group were decreased（P<0.05）,and the protein expressions of Bax,Caspase3 and Caspase9 were increased（P<0.05）,in the Shenqi Fuzheng Injection group,cisplatin group,Shenqi Fuzheng injection+cisplatin group.There were no significant changes in pathway PI3K,AKT and mTOR proteins of the PI3K/AKT/mTOR signaling pathway（P>0.05）,p-PI3K,p-AKT,and p-mTOR protein expression decreased,the protein expression of PI3K,p-AKT and p-mTOR in each group was significantly down-regulated（P<0.05）.Compared with the Shenqi Fuzheng Injection group,the expression of proliferation proteins and signaling pathway proteins in the Shenqi Fuzheng Injection+Cisplatin group were significantly reduced,and the pro-apoptotic proteins were significantly increased,the difference is statistically significant（P<0.05）.Conclusions:（1）Shenqi Fuzheng Injection combined with chemotherapy can improve the clinical efficacy of gastric cancer patients,improve the quality of life,increase CD3⁺and CD4⁺T cell levels,and reduce thrombocytopenia,leukopenia,renal function damage,neurotoxic nausea and vomiting and other chemotherapy-induced the occurrence of adverse reactions.（2）AKT1 is one of the core targets of Shenqi Fuzheng Injection in the treatment of gastric cancer.AKT1 has the strongest binding force with key compounds.The PI3K/AKT/mTOR（AKT1）signaling pathway may be the core pathway of Shenqi Fuzheng Injection in the treatment of gastric cancer.（3）In vitro experimental studies have shown that Shenqi Fuzheng Injection can down-regulate PCNA and Ki67 proliferation protein of gastric cancer cells,up-regulate the expression of Bax,Caspase-3 and Caspase-9 pro-apoptotic proteins,and down-regulate the expression of Bcl-2 anti-apoptotic protein.Shenqi Fuzheng Injection can inhibit the proliferation of gastric cancer cells and promote the apoptosis of gastric cancer cells by inhibiting the PI3K/AKT/mTOR signaling pathway.（4）In vivo experimental studies have shown that Shenqi Fuzheng Injection has the effect of inhibiting tumor growth and gastric cancer growth.By inhibiting the PI3K/AKT/mTOR signaling pathway,it inhibits the proliferation of gastric cancer cells and promotes the apoptosis of gastric cancer cells,exerting the traditional Chinese medicine effect of strengthening the body to expel evil.The combination of Shenqi Fuzheng Injection and cisplatin has a synergistic effect.